Larrea tridentate, also known as Larrea divaricata, Larrea, chaparral, or creosote bush, is a shrubby plant which dominates some areas of the desert southwest in the United States and Northern Mexico as well as some desert areas of Argentina. Tea made from the leaves of Larrea tridentate has long been used in folk medicine to treat digestive disorders, rheumatism, venereal disease, sores, bronchitis, chicken pox, and the common cold.
According to Masayuki Sakakibara, et al., Flavonoid Methyl Ethers on the External Leaf Surface of Larrea Tridentate and L. Divaricata in PHYTOCHEMISTRY, vol. 15, pp. 727-731 (Pergamon Press 1976), the disclosure of which is incorporated herein by reference, the natural products on the surface of the Larrea tridentate leaves, the leaf resin, constitutes approximately 10-15% of the dry weight of the leaves and is composed of approximately 50% nordihydroguaiaretic acid ("NDGA") and related lignans, and 50% flavonoids. NDGA, extracted from Larrea tridentate by an alkaline extraction method (U.S. Pat. No. 2,382,475, incorporated herein by reference) and produced synthetically (U.S. Pat. No. 2,644,822, incorporated herein by reference) was used as an antioxidant in edible fats, butter, oils and oleaginous materials (U.S. Pat. No. 2,373,192, incorporated herein by reference), until the GRAS (Generally Recognized As Safe) status of NDGA was revoked after animal studies revealed evidence of kidney toxicity resulting from the ingestion of NDGA.
NDGA is known as a powerful antioxidant compound. However, NDGA can itself be oxidized to toxic oxidation products by chemical means or by oxidation during processing and storage. A highly reactive and toxic oxidation product of NDGA is nordihydroguaiaretic acid ortho di-a-b-unsaturated quinone ("NDGA quinone"), which according to T. J. Mabry et al., The Natural Products Chemistry of Larrea in CREOSOTE BUSH: BIOLOGY AND CHEMISTRY OF LARREA IN THE NEW WORLD DESERTS, ch. 5, pp. 115-133 (Dowden, Hutchinson and Ross, Pennsylvania 1977) (incorporated herein by reference), occurs in Larrea and Larrea extracts and probably serves as a toxin to protect the plant from being eaten by herbivores. According to a recent report by FDA scientists (W. R. Obermeyer et al., Chemical Studies of Phytoestrogens and Related Compounds in Dietary Supplements: Flax and Chaparral, 208 PROC. SOC. EXP. BIOL. MED., pp. 6-12 (1995), the disclosure of which is incorporated herein by reference), NDGA quinone is found in chaparral (Larrea tridentate) and is suspected to be a causative agent of the toxic effects associated with consumption of chaparral products.
NDGA is the dominant lignan present in Larrea tridentate. NDGA is known to possess a variety of biological effects including anti-tumor activity, enzyme inhibition activity and antimicrobial activity according to W. Donald MacRae & G. H. Neil Towers, Biological Activities of Lignans, PHYTOCHEMISTRY, vol. 23, pp. 1207-1220 (Pergamon Press 1984) (incorporated herein by reference). Additionally, NDGA and other antioxidants have been shown to be potent inhibitors of the human immunodeficiency virus type 1 (HIV) transcription. The mode of action of this anti-HIV activity was suggested to be due to the potent antioxidant activity of NDGA inhibiting a redox regulated signal transduction pathway leading to production of HIV virus.
More recently, three scientific articles, John N. Gnabre et al., Inhibition of human immunodeficiency virus type 1 transcription and replication by DNA sequence-selective plant lignans, PROC. NATL. ACAD. SCI., USA, vol. 92, pp. 11239-11243 (November 1995); John Gnabre et al., Characterization of Anti-HIV Lignans from Larrea tridentate, TETRAHEDRON, vol. 51, pp. 12203-12210 (1995); and John Noel Gnabre et al., Isolation of anti-HIV-1 lignans from Larrea tridentate by counter-current chromatography, JOURNAL OF CHROMATOGRAPHY A, vol. 719, pp. 353-364 (1996), the disclosure of all three articles being incorporated herein by reference, demonstrate that at least two lignans isolated from Larrea tridentate, NDGA and 3-O-methyl nordihydroguaiaretic acid ("Mal. 4") inhibit transcription and replication of human immunodeficiency virus type 1 by a novel mechanism. This elucidated mechanism of anti-HIV activity is thought to be due to the ability of the two identified Larrea tridentate lignans, NDGA and Mal. 4, to interfere with the binding of the Sp1 protein to Sp1 binding sites in the HIV long terminal repeat (HIV-LTR). According to this theory, by inhibiting Sp1 binding in the HIV-LTR, the promoter activity of the HIV-LTR is eliminated so that HIV transcription, HIV Tat-regulated transactivation and HIV replication do not occur. It is further theorized by the authors that viruses other than HIV, which require binding of Sp1 protein in promoter-containing Sp1 binding sites to initiate viral replication, might also be inhibited by the anti-HIV lignans isolated from Larrea tridentate and that this class of lignans, in general, may possess a broader antiviral action of important interest.
Also, in a recent article, Anneke K. Raney & Alan McLachlan, Characterization of the Hepatitis B Virus Large Surface Antigen Promoter Spl Binding Site, VIROLOGY, vol. 208, pp. 399-404 (1995), binding sites for the transcription factor Sp1 have been identified in the DNA promoter regions of at least two important viral genes of the Hepatitis B virus (HBV) which may be involved in the coordinate regulation of HBV transcription by transcription factor Sp1.
Kaposi's Sarcoma, a cancer that frequently occurs among AIDS patients, has recently been implicated to be caused by a new herpes virus, human herpes virus-8 (HHV-8). See Roland G. Nador et al., Primary Effsion Lymphoma: A Distinct Clinicopathologic Entity Associated With the Kaposi's Sarcoma--Associated Herpes Virus, BLOOD, vol. 88, no. 2, pp. 645-656 (Jul. 15, 1996), incorporated herein by reference. See also, Matt Crenson, Kaposi's Sarcoma is tied to herpes, THE PHILADELPHIA INQUIRER, p. A4 (Jul. 31, 1996), and Lawrence K. Altman, Aids Cancer Said to Have Viral Source: Breakthrough Seen in Kaposi's Sarcoma, NEW YORK TIMES .sctn. A, p. 22 (Feb. 1, 1995), incorporated herein by reference. Antiviral therapy for immunocompromised individuals is difficult to administer, as current drugs such as acyclovir are not considered practical treatments when the immune system is compromised (New England Journal of Medicine, 1992, Vol. 327, No. 11, 782-789).
Cancerous cells are commonly treated using various forms of chemotherapy, with some degree of success in many patients. Human glioma cells are among the brain tumor cells that have been treated using chemotherapy with limited success. Human glioma cells can be derived from a glioblastoma multiforme and cultured to become a drug resistant line. However, these same cells have survived treatments of 70 ug/ml BCNU, currently, the most common chemotherapeutic agent used in the treatment of glioblastoma multiforme.
Estrogens are known to promote the growth of breast cancers. Therefore, inhibiting the production of estrogens through the use of aromatase inhibitors is an important pharmaceutical accomplishment toward the treatment and/or prevention of breast cancers. Cancer Research 53, 4563-4566, Oct. 1, 1993. Lignans and flavonoids have been shown to be competitive inhibitors of human preadipocyte aromatase. Certain naturally-occurring lignans and flavonoids have been shown to be moderate inhibitors of aromatase with K; values of between 1.3 and 27.2 micromolar concentration. These natural compounds, while not as powerful as some synthetic aromatase inhibitors, are similar in potency to some pharmaceutically useful aromatase inhibitors such as glutethimide, which has a K.sub.i value of 0.5 micromolar concentration. Aromatase functions in the conversion of androstene and testosterone to estrogens. In this capacity, lignans and flavonoids function as competitive inhibitors for the binding of the aromatase substrates, androstene and testosterone to the active site of the aromatase enzyme. Inhibition of aromatase enzyme activity, resulting in reduction of estrogen synthesis and subsequent decrease in circulating plasma estrogen levels, is thought to contribute to the prevention of estrogen-dependent cancers, such as breast cancer. (J. Steroid Biochem. Molec. Biol. Vol. 50, No. 3/4, pp. 205-212, 1994; J. Steroid Biochem. Molec. Biol. Vol. 57, No. 3/4, pp. 215-223, 1996; J. Steroid Biochem. Molec. Biol. Vol. 37, No. 2, pp. 257-260, 1990.)
Herpes zoster, also known as shingles, afflicts approximately 300,000 to 500,000 people each year in the United States. Herpes zoster most often affects elderly and immunocompromised patients, such as those with AIDS, cancer, infections, physical trauma or those undergoing certain drug therapies. It is estimated that zoster will occur at some time in ten to 20 percent of the population. Men and women are affected equally by zoster and there are no apparent seasonal variations or racial predilections. Herpes zoster occurs at a constant rate of two to three cases per 1,000 between 50 and 80 years and increases again to 10 cases per 1,000 in persons over 80 years of age.
The etiological role of varicella-zoster virus in herpes zoster, which occurs predominantly in persons over the age of 20, was confirmed in 1984 by molecular biology methods. Herpes zoster occurs due to the reactivation of the varicella-zoster virus that has been dormant in the basal root ganglia. Studies of acute herpes zoster have shown that the dorsal root ganglia sustain the brunt of the damage due to viral reactivation and replication. The damage is accompanied by localized acute inflammation, tissue necrosis and often hemorrhaging. In more severe cases, inflammation can spread to adjacent motor and sensory roots. In exceptional cases, inflammation may spread to anterior and posterior horns or posterior columns.
Herpes zoster is usually present as a painful, unilateral vesicular eruption within a single dermatotome. In young, immunocompetent individuals, the course of zoster is usually benign. In elderly patients, however, acute neuritis and postherpetic neuralgia (PHN) may develop. Severe cases of zoster and PHN can be debilitating. Immunocompromised persons are at risk for more serious disease and increased morbidity. PHN is usually defined as pain persisting for more than a month after the date of the original zoster eruption. Overall, about 10% of patients presenting with an acute case of herpes zoster will subsequently get PHN. A third of patients with PHN will still have pain persisting a year or more later. The development and persistence of PHN is greatly increased in elderly patients; almost 50% of those over 70 years may be so afflicted. (The New England Journal of Medicine, 1994, Vol. 330, No. 13, p. 896-900; afp Practical Therapeutics, 1991, Vol. 44, No. 1, p. 203-210). Postherpetic neuralgia is most commonly associated with herpes zoster although neuralgia may be associated with other types of herpes virus infections.
Besides herpes zoster, other members of the herpes virus family that may cause human disease include herpes simplex 1 and herpes simplex 2, cytomegalovirus (CMV), Epstein-Barr virus, human herpes virus 6, and human herpes virus 8. New members of the human herpes virus class continue to be discovered and undoubtedly many more exist in the global population. Recently, the inflammatory process associated with Alzheimer's disease has been linked with the presence of herpes simplex virus in the brain of susceptible individuals (The Lancet, 1997, Vol. 349, p. 241-244). Human herpes virus 6 has recently been linked to multiple sclerosis (Proceedings of the National Academy of Sciences, Vol. 92, p. 7440-7444; National Multiple Sclerosis Society Research Highlights, Summer/Fall 1997, p. 4-6). Cytomegalovirus (CMV) has been linked with the inflammatory process which may lead to hypertension, arteriosclerosis, atherosclerosis and coronary artery disease. Epstein-Barr virus has been linked with human cancers including, Burkitt's lymphoma, naspharyngeal cancer and B-lymphomas (Science, 1991, Vol. 254, p. 1167-1173).
For diseases caused by herpes virus infections including, herpes zoster, herpes simplex and Kaposi's sarcoma, antiviral agents such as acyclovir, valciclover, famiciclover, cytarabine, idoxuridine, and vidarabine may be indicated (The New England Journal of Medicine, 1992, Vol. 327, No. 11, p. 782-789; afp Practical Therapeutics, 1991, Vol. 44, No. 1, p. 203-210; The Lancet, 1990, Vol. 336, p. 537-538; Emergency Medicine, April 1997, 125-126.) The primary action of these drugs is to inhibit viral DNA synthesis. Vidarabine and acyclovir are both approved by the U.S. Food and Drug Administration for the treatment of zoster. Most clinicians consider acyclovir to be the drug of choice because of its relatively low toxicity. Acyclovir has been shown to accelerate lesions healing and lessen the pain of the infection's acute stage.
The treatment goals in patients with herpes zoster are to minimize discomfort, shorten the duration of symptoms, prevent dissemination and other complications, and prevent or minimize postherpetic neuralgia (afp Practical Therapeutics, 1991, Vol. 44, No. 1, p. 203-210). Many currently used antiviral medications for herpes virus infections, including acyclovir, do not appear to significantly reduce the incidence of duration of postherpetic neuralgia, which can occur in relatively healthy and immonocompetent elderly persons (The New England Journal of Medicine, 1992, Vol. 327, No. 11, p. 782-789; The Lancet, 1990, Vol. 336, p. 537-538). Recently, it has been demonstrated that corticosteroids (i.e. prednisolone or prednisone), which function as anti-inflammatory agents, in combination with antiviral therapy, can resolve episodes of acute pain and improve quality of life for the elderly immonocompetent patient (The Lancet, 1990, Vol. 336, p. 537-538; Emergency Medicine, April 1997, p. 125-126; Annals of Internal Medicine, 1996, Vol. 125, p. 376-383; Annals of Internal Medicine, 1997, Vol. 126, p. 831-832).
Besides the combination of acyclovir and corticosteroids, other recognized preventions and treatments for postherpetic neuralgia include: tricyclic anti-depressant compounds (i.e. amitirptylene; possibly combined with a neuroleptic agent, i.e. a phenothiazine compound), anticonvulsant compounds (i.e. carbamazepine), neuroaugmentation with topical ethyl chloride spray to control pain, a substance P antagonist (i.e. topically applied capsaicin), transcutaneous nerve stimulation, strong analgesics and narcotics (i.e. acetominophen, codeine), nonsteroidal anti-inflammatory agents (i.e. ibuprofen), topical anti-inflammatory agents (i.e. benzydamine), short courses of corticosteroid therapy (i.e. trimacinolone, prednisolone, prednisone), nerve blocks (i.e. lidocaine, bupivacaine, procaine; possibly combined with a steroid compound or noradrenaline), topical local anesthetics (i.e. Eutectic Mixture of Local Anesthetics) and surgical procedures. It has been noted in the medical literature that no treatment for PHN has proven to be uniformly successful, although some treatments may offer significant benefits. (The Lancet, 1990, Vol. 336, p. 537-538;; afp Practical Therapeutics, 1991, Vol. 44, No. 1, p. 203-210; Herpes Zoster and Post Herpetic Neuralgia, C. P. N. Watson, ed., 1993, Elsevier Science Publishers B. V., Amsterdam, Chapters 12-15).
In addition to corticosteroids, many other anti-inflammatory agents have been identified and are in common use. These include, nonsteroidal anti-inflammatory drugs (NSAIDs), and substances containing certain naturally occurring, anti-inflammatory fatty acids such as gamma-linolenic acid, alpha-linolenic acid and omega-3-fatty acids.
The class of nonsteroidal anti-inflammatory drugs include aspirin, ibuprofen, phenylbutazone and indomethacin as well as others. All the commonly used NSAIDs exert their analgesic and anti-inflammatory effects, at least in part, by inhibition of cyclooxygenase, the enzyme that initiates transformation of arachidonic acid to prostaglandins, prostacyclin and thromboxanes. Currently used NSAIDs are generally safe, well tolerated and effective when properly administered. NSAIDs are widely used for treatment of pain and inflammation and are often formulated with other pharmaceutical agents, such as caffeine or opioids, to achieve specific pharmaceutical effects. Formulations containing NSAIDs are represented in both prescription and over-the-counter drugs.
The anti-inflammatory effects of certain naturally occurring fatty acids, especially the omega-3-fatty acids and gamma-linolenic acid (GLA), are now widely known. (Proceedings of the Nutritional Society, 1996, Vol. 55, p. 737-775; Annals of Nutrition and Metabolism, 1996, Vol. 40, p. 175-182). Arachidonic acid (AA) derived eicosanoids are involved in mediating inflammatory responses. Since omega-3-fatty acids diminish the production of eicosanoid mediators, these fatty acids exert anti-inflammatory activity. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), which are abundant in fish oils and some marine plant oils, have been shown in numerous studies to exert anti-inflammatory effects. Additionally, the omega-3-fatty acid, alpha-linolenic acid (LNA), which is abundant in some vegetable oils, including flax seed and perilla seed oils, has been shown to possess anti-inflammatory activity.
Gamma-linolenic acid (GLA) is not an omega-3 fatty acid, but rather, it is an omega-6 fatty acid like arachidonic acid (AA). Most, but not all eicosanoids potentiate the inflammatory pathway which leads to inflammatory diseases. The "1-" series prostaglandins produced by the metabolism of gamma-linolenic acid in place of arachidonic acid actually possess anti-inflammatory properties. Additionally, GLA also inhibits the production of inflammatory eicosanoids by competing with arachidonic acid for eicosanoid synthesis enzymes. A particularly rich source of gamma-linolenic acid is evening primrose oil.
Capsaicin, a natural product extracted from hot peppers of the botanical genus Capsicum, has been used in the treatment of postherpetic neuralgia, other types of painful nerve disorders and inflammatory conditions. Hot peppers and their extractives have been used in food products and herbal medicine since ancient times. Extractives of Capsicum peppers contain differing amounts of capsaicin depending on the variety of pepper extracted and the extraction conditions employed. Capsaicin does not necessarily need to be purified from the other components of hot pepper extracts in order to be useful in pharmaceutical preparations. Hot pepper extracts including oleoresins and alcoholic extracts have been used in herbal medicine. The warming or burning sensation evoked by the topical application of capsaicin or an extract of Capsicum peppers may be partially masked by pretreatment or simultaneous application of a topical anesthetic (i.e. lidocaine).
There are many topical anesthetics to choose from which may be formulated with natural or synthetic antiviral agents for treatment of herpes virus infections and the pain associated with them. Two topical anesthetics commonly used in over-the-counter preparations are benzocaine and lidocaine. A naturally occurring topical anesthetic, cocaine, could also be used in place of the synthetic anesthetic compounds although, given the bad reputation associated with widespread abuse of cocaine, use of this compound is questionable in actual practice.
The combination of an effective antiviral therapy with another type of therapy to control the pain and/or inflammation associated with herpes virus infections, such as PHN, has great utility and represents an additional treatment option where, currently, effective treatment options are limited.
Certain flavonoid compounds, especially members of the chemical classes flavones and flavonols can inhibit HIV activation at fairly low concentrations (See , J. William Critchfield et al., Inhibition of HIV Activation in Latently Infected Cells by Flavonoid Compounds" in AIDS Research and Human Retroviruses, AIDS RESEARCH AND HUMAN RETROVIRUSES, Vol. 12, no. 1, pp. 3946 (1996), incorporated herein by reference). As further cited in Masayuki Sakakibara, et al., Flavonoid Methyl Ethers on the External Leaf Surface of Larrea Tridentate and L. Divaricata in PHYTOCHEMISTRY, vol. 15, pp. 727-731 (Pergamon Press 1976), Lariea tridentata contains an abundance of these classes of antiviral flavonoids, particularly methyl ethers of flavonols.
Like many physiologically active chemicals isolated from plant sources, these antiviral lignans and flavonoid compounds appear to work synergistically with other unresolved compounds present in crude extracts of Larrea tridentate. The identity and mode of action of these synergistic compounds is unknown but they may facilitate absorption of the antiviral lignans or otherwise enhance the specific physiological, antiviral effects.
NDGA is known to be a potent inhibitor of the enzyme 5-lipoxygenase. One of the enzymatic products of 5-lipoxygenase is 5-hydroperoxyeicosatetraenoic acid (HPETE) which is the precursor compound for the biosynthesis of very potent chemical mediators of inflammation, known as leukotrienes. As detailed in William R. Henderson, The Role of Leukotrienes in Inflammation, ANN. INTER. MED., vol. 121, pp. 684-697 (1994), the disclosure of which is incorporated herein by reference, 5-lipoxygenase is limited to a specific number of myeloid cells including: neutrophils, eosinophils, monocytes, macrophages, mast cells, basophils and B-lymphocytes. Leukotrienes are chemicals which induce prolonged muscle contraction, especially in the bronchioles of the lungs, and also increase vascular permeability and attract neutrophils and eosinophils to the site of inflammation. The leukotrienes play a major role in the inflammatory response to injury. Leukotrienes have also been implicated in the pathogenesis of several inflammatory diseases including: asthma, psoriasis, rheumatoid arthritis and inflammatory bowel disease. The role of leukotrienes as mediators of inflammatory diseases makes them attractive targets for therapeutic drugs to treat these diseases.
Many inhibitors of leukotriene synthesis are being developed. Recently, a 5-lipoxygenase inhibitor, Zileuton, was found to be effective in the treatment of asthma during clinical tests (Elliot Israel et al., Effect of Treatment With Zileuton, a 5-Lipoxygenase Inhibitor, in Patients With Asthma, JAMA, vol. 275, pp. 931-936 (Mar. 27, 1996), the disclosure of which is incorporated herein by reference). The success of Zileuton underscores the utility and need of therapeutic agents containing 5-lipoxygenase inhibitors in the treatment of inflammatory disease processes, including asthma.
Throughout this specification and claims, viral diseases are intended to include all diseases, attributed to a pathological virus of humans or animals, in which the causative viral agent which requires the Spl class of proteins to initiate viral replication, including certain viral agents of venereal diseases such as the Herpes viruses (the Herpesviridae), HSV-1 and HSV-2, viral hepatitis (the Hepadnaviridae) such as hepatitis B, and members of the retrovirus family (the retroviridae) including Varicella-Zoster viruses, cytomegalovirus (CMV), the human T-lymphotrophic viruses 1 and 2 (HTLV-1 and (HTLV-2) the human immunodeficiency viruses 1 and 2 (HIV-1 and HIV-2) and the cancer Kaposi's Sarcoma. Inflammatory diseases, throughout the specification and claims, are intended to include all diseases in which leukotrienes are known to play a major role or have been implicated including: asthma, allergic rhinitis, psoriasis, rheumatoid arthritis, inflammatory bowel disease, inflammatory pain, cystic fibrosis, adult respiratory distress syndrome, glomerulonephritis, inflammation of the skin, and virally induced inflammation (caused by CMV and other members of the Herpesviridae) leading to atherosclerosis/ arteriosclerosis and subsequent coronary artery disease.
In light of the foregoing. background, there exists the need for a commercial method of producing a Larrea tridentate extract which contains a high concentration of both the identified antiviral lignans (NDGA and Mal. 4), flavonoids, and a wide variety of other associated organic compounds from the leaf resin, which may contribute synergistic antiviral and lipoxygenase inhibitory activity.
Additionally, for the purpose of toxicological safety, it is of critical importance that the Larrea tridentate extract, to be used for medical applications, be processed to reduce the concentration of the toxic compound, NDGA quinone, which is reported to occur naturally in Larrea tridentate plant tissues. There is also a need to inhibit the natural production of toxic oxidation products, such as NDGA quinone, in the Larrea tridentata extract during processing and storage of the extract and formulated products and to facilitate the processing of the concentrated extract as either a liquid, slurry, or solid.
Lastly, there is a need for products comprising synthesized NDGA which is stabilized against oxidation into NDGA quinone during processing and storage. Specifically, there exists the need for additional, effective pharmaceutical treatment products for cancer cells, which may include human glioma cells. There is also an existing need for a natural, non-toxic chemical composition formulated from a nontoxic extract of Larrea tridentate which would have significant advantages over synthetic chemicals that are used to treat breast cancer. There also exists the need for additional, effective pharmaceutical treatment products for herpes virus infections, which may include pronounced inflammatory reactions, such as postherpetic neuralgia (PHN). Additional value may be added if a combination of antiviral therapy and therapy to control the pain and inflammation associated with herpes viruses could be formulated into convenient topical and oral usage forms. Still, additional value may be added if one or more components of the pharmaceutical composition are naturally occurring substances since natural compounds may offer cost savings, increased customer acceptance and more rapid progression through the testing and marketing process.